The Role And Mechanism Of Ccr2 Gene For Macrophages Mediating Renal Lymphangiogenesis

Backgroud and ObjectivesLymphangiogenesis,the development and proliferation of lymphatic vessels?LVs?,is a necessary physical process in embryonic development.And lymphangiogenesis also occurs under different pathological conditions such as tumor metastasis,inflammation,wound healing and transplant rejection.Recent studies have demonstrated that inflammation-associated lymphangiogenesis also occurs in chronic kidney disease and renal fibrosis induced in animal models of unilateral ureteral obstruction?UUO?.Macrophages are considered as the most important inflammatory cells contributing to lymphangiogenesis.However,the mechanism for macrophages to regulate renal lymphangiogenesis is unclarified.In our study,we focus on the C-C motif chemokine receptor 2?CCR2?,which is mainly expressed on monocytes/macrophages,to detect the specific mechanism for macrophages mediating renal lymphangiogenesis.Methods and ResultsFirstly,the Ccr2 knockout(Ccr2^-/-)and wild-type?WT?C57BL/6 mice were introduced to unilateral ureteral obstruction?UUO?operation for 5 days or 14 days.The Periodic Acid-Schiff?PAS?,Masson's trichrome staining and western blot results revealed that loss of Ccr2 significantly attenuates UUO-induced renal injury and fibrosis.The immunofluorescence results of LYVE-1?a specific marker for lymphatic vessels in adult mice?and Ki67?a cell proliferation marker?indicated that the numbers of lymphatic vessels and proliferating lymphatic endothelial cells in UUO-induced Ccr2^-/-kidneys were both decreased compared with that of WT mice.The above results demonstrated that loss of Ccr2 could also inhibit renal lymphangiogenesis on the basis of attenuating renal injury and fibrosis.Next,we examined the expression of vascular endothelial growth factor C?VEGF-C?,an essential growth factor required for lymphangiogenesis.The western blot and RT-PCR results revealed that the protein and mRNA levels of VEGF-C in UUO-induced Ccr2^-/-kidneys were both lower than that of WT mice.We then conducted immunohistochemical analysis of serial UUO-induced renal sections by staining VEGF-C and Aquaporin 1,a marker for proximal tubular epithelial cells?TECs?.VEGF-C was noted to be expressed by most of TECs.However,loss of Ccr2 did not affect the expression levels of VEGF-C by TECs following days 5 or 14 of UUO induction.Moreover,immunohistochemical analysis results indicated that the number of infiltrated F4/80⁺ macrophages in UUO-induced Ccr2^-/-kidneys was significantly less than that of WT mice.And double immunofluorescence results demonstrated that F4/80⁺ macrophages expressed high levels of VEGF-C.However,fewer VEGF-C-expressing F4/80⁺ macrophages and lower levels of VEGF-C expression in those macrophages were observed in the kidneys derived from Ccr2^-/-mice following days 5 and 14 of UUO induction.It demonstrated that Ccr2 deficiency attenuates VEGF-C expression in a manner independent of TECs following UUO induction,while which correlates closely with the decreased infiltrated macrophages.The immunohistochemical analysis of serial UUO-induced renal sections the F4/80+/VEGF-C+ macrophages were co-localized with the LYVE-1+ LVs,suggesting that the infiltrated macrophages probably generate a microenvironment in favor of lymphangiogenesis by expressing VEGF-C.Next,immunofluorescence results of LYVE-1 demonstrated depletion of macrophages significantly attenuated UUO-induced lymphangiogenesis.Moreover,the immunofluorescence and western blot results revealed that adoptive transfer of wild type bone marrow derived macrophages?WT BMDMs?to Ccr2^-/-kidneys could restore the capability of Ccr2^-/-mice for induction of renal lymphangiogenesis and VEGF-C expression.In the further molecular mechanisms research,we checked with focus on the PI3K-AKT-mTOR signaling pathway and hypoxia inducible factor la?HIF-1??by western blot.And the levels of phosphorylated PI3K/AKT/mTOR and HIF-1? were all increased.But the levels of above proteins were lower in Ccr2^-/-kidneys than that of WT mice.Next,the EMSA result revealed that the DNA binding activity of HIF-1? in Ccr2^-/-kidneys was weaker than that of WT kidneys.We found HIF-1? was expressed in nuclear of most macrophages.The chromatin immunoprecipitation result indicated that HIF-1? could directly bind to the promoter of VEGF-C at-498bp site.VEGF-C promoter luciferase reporter gene result further confirmed that HIF-1? could bind to the promoter of VEGF-C and promote the transcription of VEGF-C.Then WT and Ccr2^-/-BMDMs were prepared and then subjected to monocyte chemoattractant protein 1?MCP-1?stimulation.As expected,the levels for the phosphorylated PI3Kp85/AKT/mTOR and HIF-1? as well as VEGF-C expression were significantly increased in MCP-1 stimulated WT BMDMs,while no significant change was detected in MCP-1 stimulated Ccr2^-/-BMDMs.In line with these results,treatment of WT BMDMs with wortmannin?a PI3K inhibitor?,or perifosine?an AKT inhibitor?,or rapamycin?an mTOR inhibitor?,or 2-MeOE2?a HIF-1? inhibitor?significantly inhibited MCP-1 stimulated VEGF-C expression.However,LB-100?a protein phosphatase-2A inhibitor?significantly enhanced PI3Kp85/AKT/mTOR signaling along with increased HIF-1? and VEGF-C expression.Altogether,these results confirmed that CCR2.mediates UUO-induced renal lymphangiogenesis by regulating PI3Kp85-AKT-mTOR signaling and HIF-1?/VEGF-C expression.ConclusionIn conclusion,we demonstrated experimental evidence indicating that macrophages play a critical role in UUO-induced renal lymphangiogenesis,while the expression of CCR2 on macrophages is required for UUO-induced renal lymphangiogenic processes.Our studies characterized a novel regulatory network in macrophages,in which CCR2 is required to activate PI3K-AKT-mTOR signaling,which then enhances its downstream gene HIF-1?expression,and by which HIF-la directly binds to the VEGF-C promoter to transcribe its expression,thereby enhancing UUO-induced renal lymphangiogenesis.